mRNA-Seq of mayfly (Cloeon dipterum) exposed to different Fipronil concentrations below acute toxicity levels against untreated controls
mRNA-Seq of mayfly (Cloeon dipterum) exposed to different Fipronil concentrations below acute toxicity levels against untreated controls
Three (3) emerged female imagines were randomly selected and carefully transferred to a 1.5 ml Eppendorf tube for each sample. After collecting individuals for all samples, they were all euthanized at the same time by immersing the tubes in liquid nitrogen. Thereafter, 350 L of RP1 lysis buffer (NucleoSpin RNA Protein kit; Macherey-Nagel 740933.250) was added to the tube to homogenize the tissue. Prior to extraction, RP1 buffer was freshly prepared with TCEP as a reducing agent. Please see the \"Growth protocol\" for more information on incubation conditions and adult broodstock husbandry. Please see the \"Treatment protocol\" for more information on exposure duration and conditions. Cloeon dipterum is a mayfly species found in central Europe that belongs to the Baetidae (Ephemeroptera) family. Unlike the majority of mayfly larvae, C. dipterum larvae prefer lentic habitats such as small ponds, lake littorals, or slow parts of running waters. The larvae are grazers that live in macrophytes or sediment and feed on periphyton and detritus. C. dipterum has a bivoltine life cycle, with a fast-developing summer generation and a slow-developing winter generation. Fipronil (CAS:120068-37-3, PESTANAL© analytical grade) was purchased from Merck KGgA (Darmstadt, Germany). To prepare the test solution, a high concentrated stock solution in Acetone (99.9% MS grade) was first prepared. This stock solution for the highest test solution was further diluted with acetone to prepare the acetonic stock solutions for the lower test solutions. 1.00 mL of the corresponding acetonic stock solution was transferred and distributed along the glass wall of an empty glass bottle while the bottle was slowly turned. The acetone was evaporated by blowing compressed air into the bottle. The glass bottles were then filled with 2.00 L Elendt M4 medium each. The test solutions were treated with ultrasonication (30 minutes) and stirred for 60 minutes at ambient temperature before application in the test. Two chronic toxicity tests were performed per week, with a semi-static test design and two media renewals. The tests used small larvae in the L3 stage, based on Cianciara's (1976) description of wing pad development. There were at least four replicates per test concentration and control, with five individuals per replicate. The test solutions and food were changed twice a week. The tests were carried out at a temperature of 20 °C (±1 °C), with illumination of 1 E m2s 1 and a light and dark rhythm of 16:8 h. The physico-chemical parameters pH, oxygen concentration, and oxygen saturation were determined using the multiparameter device WTW Multi 1970i at the start of the test, during each media renewal, and at the end of the test. The temperature was continuously measured, and the lighting was determined once a week. C. dipterum larvae were exposed to Fipronil at nominal concentrations ranging from 0.038 to 0.60 g/L in the long-term exposure experiment. This experiment lasted 38 days, or until all of the larvae emerged. To prevent emerged imagines from escaping, the test vessels were covered with loose nets during this experiment. Once the subimagines had molted, the sex of the imagines was determined using male mayfly characteristics such as copulation tongs and turban eyes. Following that, the emerging female images were sampled for transcriptome analysis. For this, a maximum of three individuals per replicate was transferred to an Eppendorff tube containing 350 µL lysis buffer. The pooled female images in RP1 buffer were transferred to a screw-cap eppendorf tube containing 0.3 g of Lysing Matrix D ceramic beads (MP Biomedicals, 6913050). FastPrep-24 (MP Biomedicals, 6004500) was used to homogenize tissue at 5 m/s for 45 seconds at room temperature. Total RNA was extracted from the tissue lysate using the NucleoSpin RNA Protein kit (Macherey-Nagel, 740933.250) as directed by the manufacturer. A Nanodrop 2000 spectrophotometer was used to measure the concentration of RNA (>100 ng/l) (Thermo Scientific). The overall RNA quality and corresponding RIN value of the sample were determined fluorometrically using an Agilent 2100 Bioanalyzer Instrument system (Agilent, G2939BA) and the Agilent RNA 6000 Nano Kit (Agilent, 5067-1511) according to the manufacturer's protocol. Only samples with a RIN > 8 were used for downstream analysis. Samples were stored at -80°C until they were sent to the sequencing facility on dry ice. At the Eurofins Genomics GmbH sequencing facility in Germany, sequencing libraries were created from 100 ng/l total RNA for each sample. Strand-specific cDNA libraries were created using their standard workflow from protein coding mRNA molecules purified using PolyA selection.